Most cellular functions are performed by large molecular assemblies and information about the structures and functions of these higher-order complexes can lead to an improved understanding of many biological processes, including how diseases progress, at the molecular level. The goal of the proposed research is to develop an integrated approach to determining detailed information about the higher-order structures of proteins and macromolecular assemblies that combines high-resolution ion mobility spectrometry (IMS) with solution-phase chemistry and hydrogen deuterium exchange (H/DX) with supercharging tandem mass spectrometry, and apply these methods to determine detailed information about the structures and structural transitions that occur in important macromolecular complexes where only limited information has been obtained using conventional high resolution structural methods. A low-pressure drift tube apparatus will be constructed and interfaced to a QTOF mass spectrometer to obtain absolute collision cross sections of large macromolecular complexes. These cross sections can provide information about structure/function relationships of the complex, as well as provide reference measurements for others using commercially available traveling wave IMS instruments that only provide relative cross section measurements. This IMS capability will be used to develop new H/DX methods that use supercharging reagents to unfold, dissociate, and highly charge the constituent molecules in the ms or sub-ms time frame of ion formation by electrospray, which eliminates any back exchange that can occur with conventional acid quenching methods. The goal is to obtain backbone amide exchange rates with close to individual amino acid resolution using electron transfer or electron capture dissociation tandem mass spectrometry, which have been shown to minimize gas-phase H/D scrambling and the concomitant loss of exchange rate information. The effect of increased charging obtained with these reagents on any gas-phase H/D scrambling that may occur will be investigated. A proteolytic 'nicking' strategy will be pursued to significantly increase the molecular size range where this top-down method can be used. A novel method to measure assembly kinetics at short times will be developed and used to find molecular frameworks that have the potential to be developed into effective drugs for treatment of anthrax infection and proteasome inhibitors in development for cancer therapy. These integrated approaches can potentially increase the speed, sensitivity, and molecular size range for which structural information can be obtained with high fidelity, and will provide complementary information to other structural methods, including electron microscopy, NMR, crystallography, and molecular modeling. These approaches will be applied to investigate the structures and structural transitions that occur in several complexes where limited information has been obtained, including Anthrax toxin complexes, and the 26S proteasome. PUBLIC HEALTH RELEVANCE: This research is aimed at developing a fast and sensitive integrated approach for obtaining detailed information about the structure/function relationships of large molecular assemblies. This methodology can be used where conventional structural methods provide only limited information, and should be applicable to a wide range of areas of biomedical interest, including discovery of therapeutic agents aimed at regulating biomolecular complex formation, signal transduction, immune response, transcriptional regulation, protein translocation, apoptosis, and protein misfolding and aggregation that play a role in many diseases, including Alzheimer's disease, cystic fibrosis, spongiform encephalopathies (e.g., Mad Cow or Creutzfeldt Jakob disease), and even some cancers.